Diagnostic methods and gene therapy using reagents derived from the human metastasis suppressor gene KAI1

ABSTRACT

The isolation and characterization of a metastasis tumor suppressor gene KAI1 is disclosed and diagnostic methods and gene therapy approaches utilizing reagents derived from the nucleotide and deduced amino acid sequences of the KAI1 gene are provided.

FIELD OF INVENTION

This invention is in the field of cancer diagnostics and therapeutics.In particular, this invention relates to detection of alterations ofwild-type KAI1 gene sequence, KAI1 mRNA and KAI1 protein useful indetermining the presence of malignant cancer in a subject or a geneticpredisposition to malignancy in a subject. The invention further relatesto the use of gene therapy to restore the wild-type KAI1 gene product.

BACKGROUND OF INVENTION

It has been widely accepted that carcinogenesis is a multistep processinvolving genetic and epigenetic changes that dysregulate molecularcontrol of cell proliferation and differentiation. The genetic changescan include activation of proto-oncogenes and/or the inactivation oftumor suppressor genes that can initiate tumorigenesis as well as leadto the progression of tumors. For example, the tumor suppressor gene p53may be involved in late stages of colorectal carcinomas (Baker, S. J. etal., (1989) Science, 244: 217-221) and a putative metastasis suppressorgene, nm23, was found down-regulated in metastatic tumors versusnonmetastatic tumors (Steeg, P. S. et al., (1988) J. Natl. Canc. Inst.,80:200-204). In addition, the activation of ras oncogene and theamplification of N-myc have been associated with progression of humantumors such as breast carcinomas (Liu, E. et al., (1988) Oncogene3:323-327); and neuroblastomas (Brodeur, G. M. et al., (1984) Science,224:1121-1124; Schwab, M. et al., (1984) Proc. Natl. Acad. Sci. U.S.A.,81:4940-4944) but they are unlikely to be universal determinants oftumor progression (Nicolson, G. L. Bio Essays, 13:337-342 (1991).

However despite these advances in understanding the genetic changesunderlying carcinogenesis, metastasis, which is the main cause of deathfor most cancer patients (Rosenberg, S. A., Surgical Treatment ofMetastasis Cancer (Lippincott, Philadelphia Pa. 1987)), remains one ofthe most important but least understood aspects of cancer (Liotta, L. A.et al. (1991) Cell, 64:327-336; Nicolson, G. L. (1991) BioEssays,13:337-342 and Steeg, P. S. (1992) Curr. Opin. Oncol., 4:134-141).Accordingly, the isolation of metastasis tumor suppressor genes is ofgreat importance for the diagnosis and therapy of cancers.

Cell fusion studies by Ramshaw et al. ((1983) Int. J. Cancer,32:471-478) in which hybridization of non-metastatic and metastatictumor cells produced cell hybrids which are tumorigenic but no longermetastatic demonstrated the existence of metastasis suppressor genes.More recently, Ichikawa et al. (1991) Cancer Res., 51:3788-3792)demonstrated that the metastatic ability of rat prostatic cancer cellswas suppressed when fused to non-metastatic cancer cells and that thereexpression of metastasis was associated with the consistent loss of anormal rat chromosome. A subsequent study using micro-cell-mediatedchromosome transfer further mapped a putative human metastasissuppressor gene to the 11p11.2-13 region of human chromosome 11.(Ichikawa et al. (1992) Cancer Res., 52:3486-3490) In this study, theseresearchers demonstrated that a hybrid retaining human chromosome11cent-p13 showed a suppression of metastasis while hybrids retaining11cent-p11.2 did not.

In sum, the data presented in the Ichikawa et al. papers suggested thata putative suppressor gene in the p11.2-13 region of human chromosome 11may play a role in metastasis. However to date, no gene has beenidentified in this region which is a candidate metastasis suppressorgene. Thus, there is a need in the art to identify such gene(s) in thischromosome region and to determine if any such gene(s) is associatedwith metastasis.

SUMMARY OF INVENTION

The present invention relates to methods for detecting alterations ofthe wild-type KAI1 gene where detection of such alterations is useful indetermining the presence of a malignant cancer in a subject or a geneticpredisposition to malignancy in a subject. A first method for detectingalterations of the wild-type KAI1 gene comprises analyzing the DNA of asubject for mutations of the KAI1 gene. A second method for detectingalterations of the KAI1 gene comprises analyzing the RNA of a subjectfor mutations and altered expression of the mRNA product of the KAI1gene.

The present invention therefore provides nucleic acid probes fordetection of alterations of the wild-type KAI1 gene.

The present invention further provides a diagnostic kit containingpurified and isolated nucleic acid sequences useful as PCR primers inanalyzing RNA or DNA of a subject for alterations of the wild-type KAI1gene. These PCR primers can also be used to determine the nucleotidesequence of KAI1 alleles.

A third method for detecting alterations of the wild-type KAI1 genecomprises analyzing protein of a subject for alterations in theexpression of KAI1 protein.

The invention therefore relates to antibodies to the KAI1 protein and toa diagnostic kit containing antibodies to KAI1 protein useful fordetecting alterations in KAI1 protein expression in a subject.

The present invention further provides a method for supplying thewild-type KAI1 gene to a cell having altered expression of the KAI1protein, the method comprising: introducing a wild-type KAI1 gene into acell having altered expression of KAI1 protein such that the wild-typegene is expressed in the cell.

DESCRIPTION OF FIGURES

FIG. 1 shows the results of a Northern blot of mRNA from normal tissue(human prostate) and from both metastatic (AT6.1, AT6.1-11-2 andAT6.1-11-3) and non-metastatic (AT6.1-11-1*) tumor cells. 2 μg of polyA⁺ RNA per sample was loaded in each lane and the blots were hybridizedsequentially with KAI1 cDNA and rat β-actin probes. The asterisk (*)identifies the hybrid AT6.1-11-1 that contained the 11pcen-p13 regionand was suppressed in metastatic ability.

FIG. 2 shows the results of Southern blot analysis of DNA isolated fromhuman placenta, rodent cells (A9 and AT6.1) and human-rodent microcellhybrids (A9-11neo, AT6.1-11-1*, AT6.1-11-2 and AT6.1-11-3). For eachsample, 15 μg of DNA was digested with Hind III, separated on a 1.2%agarose gel and hybridized to a KAI1 cDNA probe. As in FIG. 1, theasterisk (*) identifies the hybrid AT6.1-11-1 that contained the11pcen-p13 region and was suppressed in metastatic ability.

FIG. 3 shows the nucleotide (upper line, SEQ ID NO: 19) and deducedamino acid (lower line, SEQ ID NO: 20) sequences of the KAI1 cDNA wherethe abbreviations for the amino acid residues are: A, Ala; C, Cys; D,Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M. Met; N,Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr. Thefour putative transmembrane domains are noted by a dotted underline andthe potential N-linked glycosylation sites are doubly underlined.

FIG. 4 shows the results of Northern blot analysis of 15 μg of total RNAisolated from human normal prostate tissue and from cell lines derivedfrom human metastatic prostate cancers. The blot was hybridizedsequentially with KAI1 and human β-actin probes.

FIG. 5 shows the results of Northern blot analysis of 2 μg of poly A⁺RNA prepared from the various human tissues indicated at the top of FIG.5. The blot was hybridized sequentially with KAI1 and human β-actinprobes.

FIG. 6 shows the results of a “zoo” blot of EcoRI-digested genomic DNAof the various species indicated at the top of FIG. 6. The blot washybridized with KAI1 probe.

DETAILED DESCRIPTION OF INVENTION

The present invention relates to the cloning and characterization of ametastasis suppressor gene on human chromosome 11. The nucleotide anddeduced amino acid sequences of this gene, designated KAI1 herein, areshown in FIG. 3 and in SEQ ID NOS: 19 and 20. The nucleotide sequenceshown in SEQ ID NO: 19 was cloned from a metastasis suppressed cellhybrid clone AT6.1-11-1* and represents the wild-type KAI1 sequence.

A search of the KAI1 cDNA sequence in GenBank and EMBL databasesrevealed that the KAI1 cDNA sequence is identical to three cDNA clonesfrom human lymphocytes, designated C33, R2 and IA4 by differentlaboratories (Imai, T. et al. (1992) J. Immunol., 149, 2879-2886 (1992);Fukudome, K. et al. (1992) J. Virol., 66, 1394-1401 (1992); Gaugitsch,H. W., et al. (1991) Eur. J. Immunol., 21, 377-383 (1991); Gil, M. L. etal. (1992) J. Immunol., 148, 2826-33 (1992)). C33 is associated with theinhibition of virus-induced syncytium formation (Imai, T. et al. (1992);Fukudome, K. et al. (1992)); R2 is strongly up-regulated inmitogen-activated human T cells (Gaugitsch, H. W., et al. (1991)), andIA4 is highly expressed in several B lymphocyte lines (Gil, M. L. et al.(1992)). However, none of these three clones were suggested to functionin metastasis and the function of the protein encoded by these cloneswas not known prior to the present invention.

The present invention further relates to association of alterations ofthe wild-type KAI1 gene with metastasis. Accordingly, the presentinvention relates to methods for detecting alterations of the wild-typeKAI1 gene in a subject where such methods can provide diagnostic andprognostic information. For example, since loss of expression of theKAI1 gene has been observed in metastatic prostate tumors, these aretumors in which KAI1 has a role in metastasis. Thus, detection ofalterations of the wild-type KAI1 gene in a subject may effect thecourse of treatment chosen by a clinician. In addition, since KAI1 isexpressed in all tissues tested including spleen, thymus, prostate,testes, ovary, small intestine, colon, blood leukocyte, heart, brain,placenta, lung, liver, skeletal muscle, kidney and pancreas, alterationsof the wild-type KAI1 gene may contribute to metastasis in thesetissues.

It is further understood by those of ordinary skill in the art that themethods of the present invention are applicable to any tumor in whichalterations of wild-type KAI1 occur. Moreover, the methods of detectiondisclosed in the present invention can be used prenatally to screen afetus or presymptomatically to screen a subject at risk of having cancerbased on his/her family history. For purposes of the present invention,subject means a mammal.

According to the diagnostic methods of the present invention,alterations of the wild-type KAI1 gene are detected. “Alterations of thewild-type KAI1 gene” as used throughout the specification and claimsencompasses mutations of the wild-type KAI1 gene where such mutationsinclude deletions, inversions, insertions, transversions or pointmutations of the wild-type KAI1 gene. It is believed that many mutationsfound in tumor tissues will be those leading to decreased expression ofKAI1 protein. However, mutations leading to non-functional gene productscan also lead to malignancy. It is further understood that pointmutations can occur in regulatory regions (e.g. promoter) or can disruptproper RNA processing thus leading to loss of expression of the KAI1gene products respectively.

“Alterations of the wild-type KAI1 gene” as used throughout thespecification and claims can also be detected on the basis of alteredexpression of the wild-type KAI1-specific mRNA and KAI1 protein. Thealtered expression of these KAI1 gene products may be detected as a lossor reduction in the levels of KAI1 mRNA and protein relative towild-type levels. Alternatively, the altered expression of KAI1 proteincan encompass a loss of function of the KAI1 protein. Those of ordinaryskill in the art would therefore understand that altered expression ofthe KAI1 gene products may be caused by a variety of events, includingbut not limited to, mutations of the wild-type KAI1 gene, changes in theposttranslational modification of the KAI1 protein (e.g. glycosylation)or loss of a trans-acting factor necessary for transcription of the KAI1gene.

Provided with the KAI1 cDNA and deduced amino acid sequences shown inSEQ ID NOS: 19 and 20, design of particular probes useful in detectingalterations of the wild-type KAI1 gene is well within the skill of theordinary artisan.

In one embodiment of the invention, the method for detecting alterationsof the KAI1 gene comprises analyzing the DNA of a subject for mutationsof the wild-type KAI1 gene. For analysis of DNA, a biological specimenis obtained from the subject. Examples of biological specimens that canbe obtained for use in the present methods include, but are not limitedto, tissue biopsies, whole blood, lymphocytes and tumors. Means forenriching a tissue preparation for tumor cells are known in the art. Forexample, the tissue may be isolated from paraffin or cryostat sections.Cancer cells may also be separated from normal cells by flow cytometryand other techniques well known in the art. Alternatively, primary cellcultures can be established from tumor biopsies using methods known tothose of ordinary skill in the art.

The DNA isolated from the biological specimen can be analyzed formutations of the wild-type KAI1 gene by a variety of methods including,but not limited to, Southern blotting after digestion with theappropriate restriction enzymes (restriction fragment lengthpolymorphism, RFLP) (Botstein, D. (1980) Amer. J. Hum. Genet.,69:201-205, denaturing gradient electrophoresis technique (Myers, R. M.,(1985) Nature, 313:495-498), oligonucleotide hybridization (Conner, R.et al., (1984) EMBO J., 3:13:321-1326), RNase digestion of a duplexbetween a probe RNA and the target DNA (Winter, E. et al., (1985) Proc.Natl. Acad. Sci. U.S.A., 82:7575-7579), polymerase chain reaction (PCR)(Saiki, P. K. et al., (1988) Science, 239:487-491; U.S. Pat. Nos.4,683,195 and 4,683,202), ligase chain reaction (LCR) (European PatentApplication Nos. 0,320,308 and 0,439,182), and PCR-single strandedconformation analysis (PCR-SSCP) (Orita, M. et al. (1989) Genomics,5:874-879; Dean, M. et al. (1990) Cell, 61:863-871).

In one preferred embodiment, Southern blot analysis can be used toexamine DNA isolated from a subject for gross rearrangement of the KAI1gene. The DNA to be analyzed via Southern analysis is digested with oneor more restriction enzymes. Following restriction digestion, resultantDNA fragments are separated by gel electrophoresis and the fragments aredetected by hybridization with a labelled nucleic acid probe (Southern,E. M. (1975) J. Mol. Biol., 98:503-517).

The nucleic acid sequence used as a probe in Southern analysis can belabeled in single-stranded or double-stranded form. Labelling of thenucleic acid sequence can be carried out by techniques known to oneskilled in the art. Such labelling techniques can include radiolabelsand enzymes (Sambrook, J. et al. (1989) in “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.). Inaddition, there are known non-radioactive techniques for signalamplification including methods for attaching chemical moieties topyrimidine and purine rings (Dale, R. N. K. et al. (1973) Proc. Natl.Acad. Sci., 70:2238-2242; Heck, R. F. 1968) S. Am. Chem. Soc.,90:5518-5523), methods which allow detection by chemiluminescence(Barton, S. K. et al. (1992) J. Am. Chem. Soc., 114:8736-8740) andmethods utilizing biotinylated nucleic acid probes (Johnson, T. K. etal. (1983) Anal. Biochem., 133:126-131; Erickson, P. F. et al. (1982) J.of Immunology Methods, 51:241-249; Matthaei, F. S. et al. (1986) Anal.Biochem., 157:123-128) and methods which allow detection by fluorescenceusing commercially available products. Each of the nucleic acidsequences used as a probe in Southern analysis is derived from thewild-type KAI1 gene. Preferred probes are derived from having the cDNAsequence shown in SEQ ID NO: 19.

Once the separated DNA fragments are hybridized to the labelled nucleicacid probes, the restriction digest pattern can be visualized byautoradiography and compared with the restriction digest pattern of thewild-type KAI1 gene. The presence or absence of a restriction fragmentlength polymorphism (RFLP) in the restriction pattern of the subject'sDNA relative to the wild-type restriction pattern indicates analteration of the wild-type KAI1 gene.

In another preferred embodiment, genomic DNA may be analyzed formutations in the wild-type KAI1 gene via PCR-SSCP. In this method, eachof the pair of primers selected for use in PCR are designed to hybridizewith sequences in the wild-type KAI1 gene to permit amplification andsubsequent detection of mutations in the denatured amplification productvia non-denaturing polyacrylamide gel electrophoresis. In oneembodiment, primer pairs are derived from the KAI1 cDNA sequence shownin SEQ ID NO: 19.

In another embodiment, primer pairs useful in the analysis of genomicDNA mutations of the wild-type KAI1 gene may be derived from intronicsequences which border the 5′ and 3′ ends of a given exon of the KAI1gene. Examples of primer pairs permitting specific amplification ofspecific exons of the KAI1 gene include:

SEQ ID NO: 1: AGAAGATCAAGTTGAAGAGG

SEQ ID NO: 2: GGGACCTCATTTCCTAGCTG

SEQ ID NO: 3: ATGAAACTGCTCTTGTCGG

SEQ ID NO: 4: TCAGCTCTTGGCTCCCCATT

SEQ ID NO: 5: TGGGCACGGGTTTCAGGAAAT

SEQ ID NO: 6: TGCAGAGAGCCCCAAATGCA

SEQ ID NO: 7: AGGGTGAGCCGTGAGCACAA

SEQ ID NO: 8: TGCTGAGAGTACCCAGATGC

SEQ ID NO: 9: GATGGCCACACCCACGCCC

SEQ ID NO: 10: TGCATGGAGAAGGTGCAGGC

SEQ ID NO: 11: CCTCTTGCCCACCCTGACTGA

SEQ ID NO: 12: TTCACACCATTCTCCTGCCT

where SEQ ID NOS: 1 and 2 bound exon 3; SEQ ID NOS: 3 and 4 bound exon4; SEQ ID NOS: 5 and 6 bound exon 6; SEQ ID NOS: 7 and 8 bound exon 7;SEQ ID NOS: 9 and 10 bound exon 8; and SEQ ID NOS: 11 and 12 bound exon9.

Each primer of a pair is a single-stranded oligonucleotide of about 15to about 50 base pairs in length which is complementary to a sequence atthe 3′ end of one of the strands of a double-stranded target sequence.Optimization of the amplification reaction to obtain sufficientlyspecific hybridization to the KAI1 gene sequence is well within theskill in the art and is preferably achieved by adjusting the annealingtemperature. In yet another embodiment, RNA may be analyzed formutations in the KAI1 gene by RT-PCR-SSCP. In this method, singlestranded cDNA is prepared from either total RNA or polyA⁺-enriched RNAusing reverse transcriptase. In this method, each of the pairs ofprimers selected for use in PCR of the resultant single-stranded cDNAare designed to hybridize with sequences in the KAI1 cDNA which are anappropriate distance apart (at least about 100-300 nucleotides) in thegene to permit amplification and subsequent detection of mutations inthe denatured amplification product via non-denaturing polyacrylamidegel electrophoresis. Such primer pairs can be derived from the KAI1 cDNAsequence set forth in SEQ ID NO: 19. Each pair comprises two suchprimers, complementary to sequences on each strand separated bygenerally about 100 to about 300 base pairs.

The primers of this invention can be synthesized using any of the knownmethods of oligonucleotide synthesis (e.g., the phosphodiester method ofAgarwal et al. (1972) Agnew. Chem. Int. Ed. Engl., 11:451, thephosphotriester method of Hsiung et al. (1979). Nucleic Acids Res.,6:1371, or the automated diethylphosphoramidite method of Beuacage etal. (1981). Tetrahedron Letters, 22:1859-1862), or they can be isolatedfragments of naturally occurring or cloned DNA. In addition, thoseskilled in the art would be aware that oligonucleotides can besynthesized by automated instruments sold by a variety of manufacturersor can be commercially custom ordered and prepared. In one embodiment,the primers can be derivatized to include a detectable label suitablefor detecting and/or identifying the primer extension products (e.g.,biotin, avidin, or radiolabelled dNTP's), or with a substance which aidsin the isolation of the products of amplification (e.g. biotin oravidin).

The present invention therefore provides a diagnostic kit for detectingmutations of the KAI1 gene. This diagnostic kit comprises purified andisolated nucleic acid sequences useful as hybridization probes or as PCRprimers in analyzing DNA or RNA for alterations of the wild-type KAI1gene.

In an alternative embodiment, nucleic acid probes can be selected tohybridize to mutant alleles of the KAI1 gene. These allele-specificprobes are useful to detect similar mutations in other subjects on thebasis of hybridization rather than mismatches. Where such nucleic acidprobes are primer pairs which hybridize to mutations in the KAI1 genesequence, these primer pairs can be used to amplify specific mutant genesequences present in a biological sample via PCR.

The amplification products of PCR can be detected either directly orindirectly. Direct detection of the amplification products is carriedout via labelling of primer pairs. Labels suitable for labelling theprimers of the present invention are known to one skilled in the art andinclude radioactive labels, biotin, avidin, enzymes and fluorescentmolecules. The desired labels can be incorporated into the primers priorto performing the amplification reaction. A preferred labellingprocedure utilizes radiolabelled ATP and T4 polynucleotide kinase(Sambrook, J. et al. (1989) in “Molecular Cloning, A Laboratory Manual”,Cold Spring Harbor Press, Plainview, N.Y.). Alternatively, the desiredlabel can be incorporated into the primer extension products during theamplification reaction in the form of one or more labelled dNTPs. In thepresent invention, the labelled amplified PCR products can be analyzedfor mutations of the KAI1 gene via separating the PCR products bynon-denaturing polyacrylamide gel electrophoresis, denaturingpolyacrylamide gel electrophoresis (PCR-SSCP) or via direct sequencingof the PCR-products.

In yet another embodiment, unlabelled amplification products can beanalyzed for mutations in the KAI1 disease gene via hybridization withnucleic acid probes radioactively labelled or, labelled with biotin, inSouthern blots or dot blots. Nucleic acid probes useful in thisembodiment are those described earlier for Southern analysis. In afurther embodiment, detection of point mutations may be accomplished bymolecular cloning of the allele present in the tumor tissue using thecDNA sequence set forth in SEQ ID NO: 19 and sequencing that alleleusing techniques well known in the art.

A second method for detecting alterations of the wild-type KAI1 genecomprises analyzing the RNA of a subject for mutations and alteredexpression of KAI1-specific mRNA.

For the analysis of RNA by this method, RNA can be isolated from, forexample, a tumor biopsy sample obtained from said subject where saidtumors include, but are not limited to, prostate tumors.

The RNA to be analyzed can be isolated from blood or tumor biopsysamples as whole cell RNA or as poly(A)⁺ RNA. Whole cell RNA can beisolated by methods known to those skilled in the art. Such methodsinclude extraction of RNA by differential precipitation (Birnbiom, H. C.(1988) Nucleic Acids Res., 16:1487-1497), extraction of RNA by organicsolvents (Chomczynski, P. et al. (1987) Anal. Biochem., 162:156-159) andextraction of RNA with strong denaturants (Chirgwin, J. M. et al. (1979)Biochemistry, 18:5294-5299). Poly(A)⁺ RNA can be selected from wholecell RNA by affinity chromatography on oligo-d(T) columns (Aviv, H. etal. (1972) Proc. Natl. Acad. Sci., 69:1408-1412).

The methods for analyzing RNA for mutations and altered expression ofKAI1-specific mRNA include Northern blotting (Alwine, J. C. et al.(1977) Proc. Natl. Acad. Sci., 74:5350-5354), dot and slot hybridization(Kafatos, F. C. et al. (1979) Nucleic Acids Res., 7:1541-1522), filterhybridization (Hollander, M. C. et al. (1990) Biotechniques; 9:174-179),S₁ analysis (Sharp, P. A. et al., (1980) Meth. Enzymol., 65:750-768),RNase protection (Sambrook, J. et al. (1989) in “Molecular Cloning, ALaboratory Manual”, Cold Spring Harbor Press, Plainview, N.Y.),reverse-transcription polymerase chain reaction (RT-PCR) (Watson, J. D.et al. (1992) in “Recombinant DNA” Second Edition, W. H. Freeman andCompany, New York) and RT-PCR-SSCP.

Where expression of KAI1 mRNA is measured, diminished KAI1 mRNAexpression is indicative of alteration of the wild-type KAI1 gene. Onepreferred method for measuring alterations in the level of KAI1-specificmRNA expression is Northern blotting where the nucleic acid sequenceused as a probe for detecting KAI1-specific mRNA expression iscomplementary to all or part of the KAI1 cDNA sequence shown in SEQ IDNO: 19.

A second preferred method for measuring, alterations in the level ofKAI1-specific mRNA expression is detection of KAI1 mRNA expression viahybridization of a nucleic acid probe derived from KAI1 cDNA sequence toRT-PCR products generated from RNA isolated from a biological sample.

A third method for detecting alterations of the wild-type KAI1 genecomprises analyzing the protein of a subject for alteration of wild-typeKAI1 protein. In one embodiment, alteration of wild-type KAI1 proteinencompasses a loss or reduction in the level of expression of KAI1protein in a biological sample.

Examples of immunoassays useful in determining the level of expressionof KAI1 protein include, but are not limited to, immunoprecipitation,radioimmunoassay, Western blot assay, immunofluorescent assay, enzymeimmunoassay, chemiluminescent assay, immunohistochemical assay andenzyme-linked immunosorbent assay (ELISA). In addition, the aboveimmunoassays may be used in combination such as immunoprecipitationfollowed by Western blot. The above methods are described in Principlesand Practice of Immunoassay, Price and Newman, eds., Stochton Press,1991. Such assays may be a direct, indirect, competitive ornoncompetitive immunoassay as described in the art (Oelbrick, N. (1984)J. Clin. Chem. Clin. Biochem., 22:895-904). The protein to be analyzedby such methods may be obtained from biological samples such as tumorbiopsies and the protein can be obtained as a crude lysate or it can befurther purified by methods known to those of ordinary skill in the artincluding immunoaffinity chromatography using antibodies to the KAI1protein (Sambrook, J. et al (1989) in “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Press, Plainview, N.Y.). Alternatively,levels of KAI1 protein may be detected by immunohistochemistry of fixedor frozen tumor sections.

For detection of KAI1 protein by immunoassay, the present inventionprovides anti-KAI1 antibodies where such antibodies may be polyclonal ormonoclonal. If polyclonal antibodies are desired, serum containingpolyclonal antibodies to KAI1 protein can be used or the polyclonalantibodies can be purified from other antigens present in the serum byimmunoaffinity chromatography. Alternatively, monoclonal antibodiesdirected against KAI1 can readily be produced by one of ordinary skilledin the art. Methods of producing monoclonal or polyclonal antibodies areknown to one of ordinary skilled in the art (Goding, J. W. (1983)monoclonal antibodies: Principles and Practice, Plodermic Press, Inc.,NY, N.Y., pp. 56-97; Hurn, B. A. L. et al. (1980) Meth. Enzymol.,70:104-141).

Suitable immunogens which may be used to produce the polyclonal ormonoclonal antibodies of the present invention include cell lysateprepared from cells transfected with a recombinant KAI1 protein,partially or substantially purified recombinant or native KAI1 protein,or peptides derived from the KAI1 amino acid sequence shown in SEQ IDNO: 20. When purification of the recombinant or native KAI1 protein isdesired, it can be accomplished by standard protein purificationprocedures known in the art which may include differentialprecipitation, molecular sieve chromatography, ion-exchangechromatography, isoelectric focusing, gel electrophoresis, affinity, andimmunoaffinity chromatography and the like. In the case ofimmunoaffinity chromatography, the recombinant protein may be purifiedby passage through a column containing a resin which has bound theretoantibodies specific for the KAI1 protein.

In a preferred embodiment, the immunogen is a recombinantly producedKAI1 protein or fragments thereof. Production of recombinant KAI1protein or a fragment thereof may be directed by a natural or syntheticnucleic acid sequence inserted into a suitable expression vector. Apreferred nucleic acid sequence is the KAI1 cDNA sequence shown in SEQID NO: 19. In one embodiment, restriction digest fragments containingthe full-length cDNA or fragments thereof containing a coding sequencefor KAI1 can be inserted into a suitable expression vector. By suitableexpression vector is meant a vector that can function in eukaryotic orprokaryotic cells and is capable of carrying and expressing a nucleicacid sequence encoding the KAI1 protein or a fragment thereof. Suchvectors and their use in producing recombinant proteins are known tothose of ordinary skill in the art (Sambrook, J. et al. (1989) in“Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Press,Plainview, N.Y.).

The immunogen of the present invention can be used in a suitable diluentsuch as saline or water, or in complete or incomplete adjuvants.Further, the immunogen may or may not be bound to a carrier to make theprotein immunogenic. Examples of such carrier molecules include but arenot limited to bovine serum albumin (BSA), keyhole limpet hemocyanin(KLH), tetanus toxoid, and the like. The immunogen can be administeredby any route appropriate for antibody production such as intravenous,intraperitoneal, intramuscular, subcutaneous, and the like. Theimmunogen may be administered once or at periodic intervals until asignificant titer of anti-KAI1 antibody is produced. The antibody may bedetected in the serum using an immunoassay.

The antibodies or antigen binding fragments may also be produced bygenetic engineering. The technology for expression of both heavy andlight chain genes in E. coli is the subject of PCT patent applications;publication number WO 901443, WO 901443, and WO 9014424 and in Huse etal. (1989) Science, 246:1275-1281.

Alternatively, anti-KAI1 antibodies can be induced by administeringanti-idiotype antibodies as immunogens. Conveniently, a purifiedanti-KAI1 antibody preparation prepared as described above is used toinduce anti-idiotype antibody in a host animal. The composition isadministered to the host animal in a suitable diluent. Followingadministration, usually repeated administration, the host producesanti-idiotype antibody. To eliminate an immunogenic response to the Fcregion, antibodies produced by the same species as the host animal canbe used or the Fc region of the administered antibodies can be removed.Following induction of anti-idiotype antibody in the host animal, serumor plasma is removed to provide an antibody composition. The compositioncan be purified as described above for anti-KAI1 antibodies, or byaffinity chromatography using anti-KAI1 antibodies bound to the affinitymatrix.

In an alternative embodiment, the antibodies of the present inventioncan be used in situ to detect KAI1 protein in cells or tissues. In oneembodiment, the antibodies are used in direct or indirectimmunofluorescence. In the direct method, anti-KAI1 antibody labelledwith a fluorescent reagent such as fluorescein isothiocyanate, rhodamineB isothiocyanate and the like is reacted directly with the KAI1 presentin cells or tissues. In the indirect method, unlabelled anti-KAI1antibody is reacted with the KAI1 protein present in cells or tissue.The unlabelled anti-KAI1 antibody is then reacted with a labelled secondantibody. The second antibody can be labelled with a fluorescent tag asdescribed above. The fluorescently labelled cells or tissues can then bedetected using techniques known to one skilled in the art such as afluorescence-activated cell sorter, light microscopy using a fluorescentlight lamp and the like. Alternatively, KAI1 protein can be detected insitu via the use of radiolabelled anti-KAI1 antibody or via the use ofan unlabelled anti-KAI1 antibody followed by a radiolabelled secondantibody reactive to the anti-KAI1 antibody.

The antibodies of the present invention may also be used toimmunoprecipitate the KAI1 protein from a mixture of proteins. The useof immunoprecipitation as a sensitive and specific technique to detectand quantitate target antigen in mixtures of proteins is well known tothose of ordinary skill in the art (see Molecular Cloning, A LaboratoryManual, 2d Edition, Maniatis, T. et al. eds. (1989) Cold Spring HarborPress).

The antibodies of the present invention may also be affixed to solidsupports for use in the isolation of KAI1 protein by immunoaffinitychromatography. Techniques for immunoaffinity chromatography are knownin the art (Harlow, E. and Lane, D. (1888) “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.)including techniques for affixing antibodies to solid supports so thatthey retain their immunoselective activity; the techniques used may bethose in which the antibodies are adsorbed to the support as well asthose in which the antibodies are covalently linked to the support.Generally, the techniques are similar to those used in covalent linkingof antigen to a solid support; however, spacer groups may be included inthe bifunctional coupling agents so that the antigen binding site of theantibody remains accessible.

The above described antibodies and antigen binding fragments thereof maybe supplied in a diagnostic kit useful for the detection of alterationsin the expression of KAI1 protein.

In a second embodiment, alteration of wild-type KAI1 protein encompassesloss of function of the KAI1 protein. The present method thereforeincludes assays useful in determining the functional status of the KAIprotein. For example, since an association between processing ofN-linked oligosaccharides and metastatic phenotype has beenwell-documented (Hakomori, S. -I. (1989) Advanc. Cancer Res.,52:257-331; Dennis, J. W., et al. (1987) Science, 236:582-585; Ishikawa,M. et al. (1988) Cancer Res., 48:665-670) it is believed thatglycosylation of the KAI1 protein is required for the protein tofunction as a metastasis suppressor. Thus, detection of a loss offunction of KAI1 protein as evidenced by an absence of glycosylation isindicative of the presence of metastatic cancer in a subject.

The present invention also relates to a gene therapy method in which anexpression vector containing a nucleic acid sequence representing thewild-type KAI1 gene is administered to a subject having a mutation ofthe KAI1 gene. A nucleic acid sequence representing wild-type KAI1 geneis that shown in SEQ ID NO: 19. Such nucleic acid sequence may beinserted into a suitable expression vector by methods known to those ofordinary skill in the art. Expression vectors suitable for producinghigh efficiency gene transfer in vivo include retroviral, adenoviral andvaccinia viral vectors.

Expression vectors containing a nucleic acid sequence representingwild-type KAI1 gene can be administered intravenously, intramuscularly,subcutaneously, intraperitoneally or orally. A preferred route ofadministration is intraperitioneally.

Any articles or patents referenced herein are incorporated by reference.The following examples are presented to illustrate various aspects ofthe invention but are in no way intended to limit the scope thereof.

EXAMPLES Materials and Methods

Cell lines: AT6.1 is a highly metastatic Dunning rat prostatic cancercell line. AT6.1-11 clones are microcell hybrids that have a portion ofhuman chromosomes 11 as the sole human genetic materials in AT6.1 cells.Microcell hybrid AT6.1-11-1* contains a fragment of human chromosome 11,cen-p13, from the centromere to region p13, and was suppressed formetastatic ability. Microcell hybrids AT6.1-11-2 and -3 have smallerfragments of human chromosome 11 from the centromere to P11.2 and werenot suppressed for metastatic ability. The characteristics and growthcondition for these cell lines have been previously described in detail(Ichikawa et al, (1992) Cancer Res., 52:3486-3490). A9-11neo is a mouseA9 cell line containing human chromosome 11pter-q23 (Koi et al, (1989)Mol Carcinog., 2:12-21).

Isolation and Sequencing of KAI1 cDNA Clone

Poly (A)⁺ RNA was isolated from exponentially growing AT6.1-11-1* cells,using a FastTrack mRNA isolation kit (Invitrogen, San Diego, Calif.).Oligo (dT) was used to prime the first strand cDNA synthesis from 5 μgof poly (A)⁺ RNA. Double-stranded cDNA was cloned into plasmid pSPORT 1vector by procedures recommended by the vendor (GIBCO BRL, Grand Island,N.Y.). Human Alu sequence primer Alu 559 (Nelson, D. L. et al (189)Proc. Natl. Acad Sci U.S.A. 86:6686-6690.) was used to amplify genomicDNA from suppressed hybrid AT6.1-11-1* and the nonsuppressed cloneAT6.1-11-2 by PCR. The multiple Alu-PCR fragments of AT6.1-11-1* werecloned into a T-tailed vector pCR1000 (Invitrogen, San Diego, Calif.).Individual clones corresponding to each fragment of Alu-PCR productswere isolated after comparing the size of these Alu-PCR products tomolecular weight markers in a agarose gel stained with ethidium bromide.Eleven fragments unique to AT6.1-11-1* were labeled by random priming(GIBCO BRL, Bethesda, Md.) and used to screen 5×10⁴ recombinants of thecDNA library under stringent wash conditions (65° C. in 0.1×SSC+0.1% SDSfor 30 min.). Five independent clones were obtained and their insertswere sequenced using the Sequenase kit (US Biochemical, Cleveland,Ohio). DNA sequences were analyzed with the GCG package (version 7.3,1993, Madison, Wis.).

RNA analysis: Cytoplasmic RNA from AT6.1, AT6.1-11-1*, -2 and -3 wereprepared from exponentially growing cells, using FastTrack mRNAisolation kit (Invitrogen, San Diego, Calif.). Other poly (A)⁺RNA andhuman multiple tissue Northern blots were purchased from Clontech (PaloAlto, Calif.). 2 μg of poly (A)⁺RNA was denatured with formamide andfractionated on a 1.2% agarose gel in formaldehyde buffer. The RNA wasthen transferred onto nylon membrane, baked in an oven at 80° C. for 90min, and then hybridized with a labeled probe in QuickHyb hybridizationsolution (Stratagene, La Jolla, Calif.) at 68° C. for 1.5 hours andwashed at 68° C. for 30 minutes in 0.1×SSC, 0.1% SDS andautoradiographed.

DNA analysis: 15 μg of genomic DNA was digested with BamHI and separatedon a 1.2% agarose gel. Following denaturation and neutralization, theDNA in the gel was transferred onto nylon membrane. The “zoo” blotcontaining EcoR1-digested genomic DNA from human, rat, mouse, dog, cow,rabbit, chicken and yeast (Zoo-blot) was purchased from Clontech (PaloAlto, Calif.). The Southern blots were further hybridized and washedunder the same conditions as described above for the Northern blots.

PCR: All PCRs in this study were carried out in 50 μl with 25 pmol ofeach primer, 10 mM Tris. HCI, 500 mM KCI, 1.5 mM MgCl₂, 0.01% gelatin,250 mM of each dNTP and 0.25 units of Taq DNA polymerase (Perkin-Elmer,Norwalk, Conn.). The initial DNA denaturation was performed at 95° C.for 5 min, followed by 35 cycles of 94° C. denaturation for 1 min, 55°C. annealing for 1 min and 720° C. extension of 4 min, with a finalextension of 72° C. for 8 min. Probes and Oligonucleotides sequences:The KAI1 probe (nucleotides 64-1094 of the KAI1 cDNA) used in Southernand Northern blot analyses was generated by PCR with primers shown asSEQ ID NO: 13 AGTCCTCCCTGCTGCTGTGTG and SEQ ID NO:14TCAGTCAGGGTGGGCAAGAGG. Human and rat β-actin probes were PCR productsgenerated by templates and primers purchased from Clontech (Palo Alto,Calif.). The primer sequences for human β-actin are shown as SEQ ID NO:15 GAGGAGCACCCCGTGCTGCTGA and SEQ ID NO: 16 CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC and the primer sequences for rat β-actinare shown as SEQ ID NO: 17 TTGTAACCAACTGGGACGATATGG and SEQ ID NO: 18GTCTTGATCTTCATGGTGCTAGG.

Example 1 Cloning of the KAI1 Gene

To clone the gene on human chromosome 11 responsible for the metastasissuppression of AT6.1 prostatic cancer cells, genomic DNA fragments fromthe p11.2-13 region were isolated using human-specific Aluelement-mediated PCR (Alu-PCR) (Nelson, D. L. et al Proc. Natl. Acad.Sci. U.S.A., 86:6686-6690) with DNAs from the metastasis suppressedmicrocell hybrid AT6.1-11-1* and the non-suppressed hybrids AT6.1-11-2and AT6.1-11-3. The Alu-PCR fragments found only in the AT6.1-11-1 DNAwere then used as probes to screen a cDNA library prepared from thesuppressed cell hybrid clone AT6.1-11-1* that contains human chromosomalregion 11cen-p13. Of five cDNA clones obtained, all were expressed inthe suppressed hybrid but not in the nonsuppressed hybrids as detectedby reverse transcription-polymerase chain reaction (RT-PCR) usingprimers derived from these cDNA sequences. Northern analysis of RNAisolated from human prostate and cell lines AT6.1, AT6.1-11-1*, -2 and-3 revealed that two of the cDNA clones detected a 2.4 hb and 4.0 kbtranscript respectively in human tissue and the suppressed AT6.1-11-1*cells. The results of a Northern blot for one such clone, designatedKAI1 for Kang Ai (Chinese for anti-cancer), are shown in FIG. 1 andclearly demonstrate that KAI1 mRNA was abundant in the metastaticsuppressed AT6.1-11-1* cells but absent from the parental AT6.1 cellsand the nonsuppressed hybrids. Therefore, the KAI1 clone was analyzedfurther.

To confirm that the KAI1 gene was isolated from the p11.2-13 region ofhuman chromosome 11 involved in metastasis suppression, Southern blotanalysis was conducted on 15 μg of genomic DNA from human placenta,rodent cells (A9 and AT6.1) and human-rodent microcell hybrids(AT6.1-11-1*, AT6,1-11-2 and AT6.1-11-3), digested with Hind III,separated on a 1.2% agarose gel and hybridized with KAI1 probe. Theresults shown in FIG. 2 demonstrate that only the cell hybrids that havethe p11.2-13 region involved in metastasis suppression (AT6.1-11-1*)have the pattern observed with normal human DNA when hybridized to KAI1probe. Fluorescence in situ hybridization of a KAI1 probe to metaphasechromosomes further localized KAI1 to the p11.2 region.

Example 2 Nucleotide And Deduced Amino Acid Sequences of the KAI1 cDNA

The nucleotide and deduced amino acid sequences of the KAI1 cDNA areshown in FIG. 3 and in SEQ ID NOS: 19 and 20. The KAI1 cDNA has a singleopen reading frame from nucleotide positions 166 to 966, predicting aprotein of 267 amino acids with a calculated molecular weight of 29,610daltons. An Alu element was present in the 3′-untranslated region of thecDNA. The predicted protein had four hydrophobic and presumablytransmembrane domains and one large extracellular hydrophilic domainwith three potential N-glycosylation sites. As noted earlier, the KAI1cDNA sequence is identical to three cDNA clones from human lymphocytes,C33, R2 and IA4.

Example 3 Determination That KAI1 Is A Metastasis Suppressor Gene

To investigate if KAI1 is the gene responsible for metastasissuppression in AT6.1-11-1*, KAI1 cDNA was subcloned into a constitutiveexpression vector and transfected into parental AT6.1 cells as follows.In brief, KAI1 cDNA was cloned into pCMVneo, in which transcription isdriven by the constitutive human cytomegalovirus promoter (Eliyahu, D.et al Proc Natl Acad Sci U.S.A., (1989) 86:8763-8767). The resultantplasmid pCMV-KAI1 was transfected into AT6.1 cells by calcium phosphateprecipitate method and the vector alone was also transfected as anegative control. Individual transfectants were isolated in selectionmedium (RPMI-1640 plus 10% fetal calf serum, 2 units/ml pen-strep and500 ug/ml neomycin). Exponentially growing untransfected AT6.1,AT6.1-11-1* and AT6.1-11-2 cells and exponentially growing vector(AT6.1VEC-1, AT6.1VEC-2 and AT6.1VEC-3) and KAI1 (AT6.1KAI-1, AT6.1KAI-2and AT6.1KAI-3) transfectants were collected by scraping and cell clumpswere broken up by gentle pipetting. The cell suspension was placed in atube and allowed to stand at room temperature for 30 min. Cells from thesupernatant suspension were collected, washed, and resuspended in coldPBS at 10⁶ cells/ml. Four-to-five-week-old male Ncr nu/nu nude mice(National Cancer Institute Animal Program, Bethesda, Md.) were injectedwith 0.1 ml of the indicated cell suspension (10⁵ cells): (the columndesignated “Clone” in Table 1) subcutaneously at sites on both the rightand left midlateral, about ¼ of the distance from the base of the skullto the base of the tail. About 6 weeks after injection, the tumors wereweighed and the lungs were inflated with Bouin's solution. Tumor foci onthe surface of lungs were scored under a dissecting microscope.Individual transfectants were analyzed for KAI1 expression and for theirability to suppress lung metastases and the results of one experimentare shown in Table 1.

TABLE 1 Mean Number^(≠) KAI1* Tumor Tumor Number of Metastasis mRNALatency⁺ Age Weight (g) Mice with Per Mouse Mean Clones Level (days)(Days) @ Excision Metastases (# mice) P AT6.1 0 4.3 27 2.58 19/19 58(32-135) AT6.1-11-1* 10 3.7 37 2.79 6/7  7 (0-9) <0.005^(§) AT6.1-11-1-20 4.2 37 2.78 6/6 26 (20-40) AT6.1VEC-1 0 4.9 43 2.32 17/17 30 (16-57)AT6.1VEC-2 0 4.0 43 3.26 17/17 30 (12-71) AT6.1VEC-3 0 5.5 43 2.57 18/1847 (15-183) AT6.1KAI-1 10  4.2 43 3.99 18/20  6 (0-14) <0.001^(∥)AT6.1KAI-2 7 4.5 41 1.79 17/19  7 (0-17) <0.001^(∥) AT6.1KAI-3 1 4.5 432.56 18/19 23 (0-36) <0.02^(∥) The data shown in this table are from alarge, age-matched cohort of “side-by-side” nude mice, with cellsinoculated at the same time. *KAI1 expression was determined by Northernblot analysis. The KAI1 signals on the Northern blot were scored by adensitometer. The value for AT6.1KAI-1 was standardized to 10 and thevalues for other clones were adjusted accordingly. ⁺Latency is the timefollowing injection for a palpable tumor to appear. ^(≠)The numbers inparentheses indicate the range of metastases in individual mice.^(§)Compared to the number of metastases with AT6.1-11-2 cells.^(∥)Compared to the mean number of metastases with all of the threevector transfectants.

The results presented show that expression of KAI1 resulted in asignificant suppression of the number of lung metastases per mouse butdid not affect the growth rate of the primary tumor. Further, whereasthe parental AT6.1 cells yielded 58 metastasis per mouse when injectedsubcutaneously into nude mice, two transfectants with levels of KAI1mRNA expression similar to the high level of expression observed inAT6.1-11-1* cells gave only 6 or 7 lung metastases per animal. Incontrast, the three vector control transfectants produced 30-47 lungmetastases per mouse, which is on average 5.5 times the number ofmetastases observed with the 2 KAI1 transfectants with high KAI1 mRNAexpression (AT6.1KAI-1 and AT6.1KAI-2). In addition, while theAT6.1KAI-3 clone which had low KAI1 expression produced 23 lungmetastases, this was still significantly less than the mean number oflung metastases for control transfectants. Finally, Northern analysisshowed that KAI1 expression was undetectable or very low in 28 lungmetastases from KAI1 transfectants suggesting that selection for cellswith absent or reduced KAI1 expression resulted in metastasis formation.These results indicate that the metastatic ability of AT6.1 cells issuppressed by KAI1 expression.

Example 4 KAI1 mRNA Expression In Cell Lines Derived From MetastaticHuman Prostate Tumors

To determine whether KAI1 mRNA expression was reduced in humanmetastatic prostate tumors relative to expression in normal humanprostate, 15 μg total RNA from human normal prostate tissue and fromcell lines derived from metastatic prostate cancers (Kaighn, M. E. et al(1979) Invest. Urol.: 17:16; Horoszewicz, J. J. et al. in Models forProstate Cancer, G. P. Murphy. Ed. (Alan R. Liss, Inc., New York, 1980).pp 115-132: T. Iizumi. et al. (1987) J. Urol. 137:1304, D. D. Mickey etal., in Models for Prostate Cancer. G. P. Murphy. Ed. (Alan R. Liss,Inc. New York, 1980), pp. 67-84) were denatured with formamide,electrophoresed fractionated on a 1.2% agarose gel and hybridizedsequentially to KAI1 and human β-actin probes. The results of thisNorthern blot analysis are shown in FIG. 4 and clearly demonstrate thatKAI1 expression was significantly reduced in the human cell linesderived from metastatic prostate tumors (PC-3, LNCaP, TSU-Pr1 and DU145)when compared to normal prostate (prostate). In addition, while longerexposures (4 days at −80 C) of the autoradiogram shown in FIG. 4(overnite at −80 C) revealed expression of KAI1 mRNA in all of the tumorcells, the level of expression was still much lower than in normalprostate.

To rule out the possibility that the metastasis suppression by KAI1 wasdue to an indirect immune mechanism, two other experiments wereperformed. First, parental AT6.1 cells, cell hybrid clone AT6.1-11-1*,or a KAI1 transfectant (AT6.1 KAI-1) were inoculated into the leg ofsevere combined immune deficient (SCID) mice at 5×10⁵ cells/mouse. Whentumors reached 3-5 cm³, the leg with tumor was surgically removed andanimals were followed until 50 to 60 days post inoculation. Lungmetastases for each mouse were analyzed as described for Table 1. ForAT6.1, 9/9 mice had lung metastases with an average number of 83 permouse. For AT6.1-11-1*, 4/9 mice had lung metastases with an averagenumber of 6 per mouse. For AT6.1KAI-1, 2/7 mice had lung metastases withan average number of 2 per mouse. These studies demonstrated that evenin SCID mice, which are more immune compromised than nude mice,metastasis suppression was observed.

Second, highly metastatic rat mammary cancer cells into which the KAI1gene was introduced via microcell-mediated chromosome transfer, retainedtheir ability to metastasize (Rinker-Schaefer, C. W. et al. (1994)Cancer Res., 54:6249-6256) even though the hybrids expressed similarlevel of KAI1 mRNA. Based upon these data, a more direct mechanismappears to be responsible for the metastasis suppression by KAI1 .Consistent with this possibility, high KAI1 expressing AT6.1-11-1*hybrid cells have about 50% reduction in their invasive ability ascompared to parental AT6.1 cells or nonsuppressed AT6.1-11-2 hybridcells in Boyden chamber assay. In brief, Boyden chamber invasion assayswere performed as described by J. Vukanovic et al. (1993) Cancer Res.,53:1833), using matrigel coated filters and 5% fetal bovine serum aschemoattractant in the lower well. During the 12 hours of the assay,19±3 parental AT6.1 cells per high power field invaded through thematrigel filters versus 10±2 for the metastasis suppressed AT6.1-11-1*hybrid cells and 18±2 for the nonsuppressed AT6.1-11-2 hybrid cells.

Example 5 Expression of KAI1 Gene In Human Tissues

To evaluate the expression level of KAI1 gene in various human tissues,Northern analysis was performed on RNA isolated from multiple humantissues. In brief, a human multiple tissue Northern blot purchased fromClontech (Palo Alto, Calif.) was hybridized sequentially with KAI1 andhuman β-actin probes under conditions described in the Methods section.The results presented in FIG. 5 show that the 2.4 kb KAI1 transcript wasdetected in all the human tissues tested, with high abundance inprostate, lung, liver, kidney, bone marrow and placenta; moderateabundance in mammary gland, pancreas, skeletal muscle and thymus; andlow expression in brain, heart, ovary, stomach and uterus.

Example 6 Conservation Of The KAI1 Gene Across Species

To determine if the KAI1 gene is evolutionarily conserved acrossspecies, a zoo blot containing EcoRI-digested genomic DNA from variousspecies was purchased from Clontech (Palo Alto, Calif.) and hybridizedwith KAI1 probe. The results presented in FIG. 6 show that theevolutionary conservation of KAI1 coding sequence is high in human,monkey, dog and rabbit and moderate in cow, rat, mouse. The evolutionaryconservation and wide tissue distribution for KAI1 suggest that the genemay have an essential biological function.

Example 7 Correlation Of Altered KAI1 Expression In Human Tissue SamplesWith Metastasis

Tumor biopsies of liver metastases from prostate cancer patients andliver biopsies from healthy patients are analyzed for KAI1 mRNAexpression by Northern blotting. KAI1 mRNA expression is lost in thetumor samples indicating that the presence of liver metastases in theprostate cancer patients is correlated with altered KAI1 expression.

20 20 base pairs nucleic acid single linear unknown 1 AGAAGATCAAGTTGAAGAGG 20 20 base pairs nucleic acid single linear unknown 2GGGACCTCAT TTCCTAGCTG 20 19 base pairs nucleic acid single linearunknown 3 ATGAAACTGC TCTTGTCGG 19 20 base pairs nucleic acid singlelinear unknown 4 TCAGCTCTTG GCTCCCCATT 20 21 base pairs nucleic acidsingle linear unknown 5 TGGGCACGGG TTTCAGGAAA T 21 20 base pairs nucleicacid single linear unknown 6 TGCAGAGAGC CCCAAATGCA 20 20 base pairsnucleic acid single linear unknown 7 AGGGTGAGCC GTGAGCACAA 20 20 basepairs nucleic acid single linear unknown 8 TGCTGAGAGT ACCCAGATGC 20 19base pairs nucleic acid single linear unknown 9 GATGGCCACA CCCACGCCC 1920 base pairs nucleic acid single linear unknown 10 TGCATGGAGAAGGTGCAGGC 20 21 base pairs nucleic acid single linear unknown 11CCTCTTGCCC ACCCTGACTGA 21 20 base pairs nucleic acid single linearunknown 12 TTCACACCAT TCTCCTGCCT 20 21 base pairs nucleic acid singlelinear unknown 13 AGTCCTCCCT GCTGCTGTGT G 21 21 base pairs nucleic acidsingle linear unknown 14 TCAGTCAGGG TGGGCAAGAG G 21 22 base pairsnucleic acid single linear unknown 15 GAGGAGCACC CCGTGCTGCT GA 22 33base pairs nucleic acid single linear unknown 16 CTAGAAGCAT TTGCGGTGGACGATGGAGGG GCC 33 24 base pairs nucleic acid single linear unknown 17TTGTAACCAA CTGGGACGAT ATGG 24 23 base pairs nucleic acid single linearunknown 18 GTCTTGATCT TCATGGTGCT AGG 23 1624 base pairs nucleic acidsingle linear unknown 19 CCGACTGAGG CACGAGCGGG TGACGCTGGG CCTGCAGCGC 40GGAGCAGAAA GCAGAACCCG CAGAGTCCTC CCTGCTGCTG 80 TGTGGACGAC ACGTGGGCACAGGCAGAAGT GGGCCCTGTG 120 ACCAGCTGCA CTGGTTTCGT GGAAGGAAGC TCCAGGACTG160 GCGGGATGGG CTCAGCCTGT ATCAAAGTCA CCAAATACTT 200 TCTCTTCCTCTTCAACTTGA TCTTCTTTAT CCTGGGCGCA 240 GTGATCCTGG GCTTCGGGGT GTGGATCCTGGCCGACAAGA 280 GCAGTTTCAT CTCTGTCCTG CAAACCTCCT CCAGCTCGCT 320TAGGATGGGG GCCTATGTCT TCATCGGCGT GGGGGCAGTC 360 ACTATGCTCA TGGGCTTCCTGGGCTGCATC GGCGCCGTCA 400 ACGAGGTCCG CTGCCTGCTG GGGCTGTACT TTGCTTTCCT440 GCTCCTGATC CTCATTGCCC AGGTGACGGC CGGGGCCCTC 480 TTCTACTTCAACATGGGCAA GCTGAAGCAG GAGATGGGCG 520 GCATCGTGAC TGAGCTCATT CGAGACTACAACAGCAGTCG 560 CGAGGACAGC CTGCAGGATG CCTGGGACTA CGTGCAGGCT 600CAGGTGAAGT GCTGCGGCTG GGTCAGCTTC TACAACTGGA 640 CAGACAACGC TGAGCTCATGAATCGCCCTG AGGTCACCTA 680 CCCCTGTTCC TGCGAAGTCA AGGGGGAAGA GGACAACAGC720 CTTTCTGTGA GGAAGGGCTT CTGCGAGGCC CCCGGCAACA 760 GGACCCAGAGTGGCAACCAC CCTGAGGACT GGCCTGTGTA 800 CCAGGAGGGC TGCATGGAGA AGGTGCAGGCGTGGCTGCAG 840 GAGAACCTGG GCATCATCCT CGGCGTGGGC GTGGGTGTGG 880CCATCATCGA GCTCCTGGGG ATGGTCCTGT CCATCTGCTT 920 GTGCCGGCAC GTCCATTCCGAAGACTACAG CAAGGTCCCC 960 AAGTACTGAG GCAGCTGCTA TCCCCATCTC CCTGCCTGGC1000 CCCCAACCTC AGGGCTCCCA GGGGTCTCCC TGGCTCCCTC 1040 CTCCAGGCCTGCCTCCCACT TCACTGCGAA GACCCTCTTG 1080 CCCACCCTGA CTGAAAGTAG GGGGCTTTCTGGGGCCTAGC 1120 GATCTCTCCT GGCCTATCCG CTGCCAGCCT TGAGCCCTGG 1160CTGTTCTGTG GTTCCTCTGC TCACCGCCCA TCAGGGTTCT 1200 CTTATCAACT CAGAGAAAAATGCTCCCCAC AGCGTCCCTG 1240 GCGCAGGTGG GCTGGACTTC TACCTGCCCT CAAGGGTGTG1280 TATATTGTAT AGGGGCAACT GTATGAAAAA TTGGGGAGGA 1320 GGGGGCCGGGCGCGGTGCTC ACGCCTGTAA TCCCAGCACT 1360 TTGGGAGGCC GAGGCGGGTG GATCACGAGGTCAGGAGATC 1400 GAGACCATCC TGGCTAACAT GGTGAAACCC CGTCTCTACT 1440AAAAATACAA AAAAAATTTA GCCGGGCGCG GTGGCGGGCA 1480 CCTGTAGTCC CAGCTACTTGGGAGGCTGAG GCAGGAGAAT 1520 GGTGTGAACC CGGGAGCGGA GGTTGCAGTG AGCTGAGATC1560 GTGCTACTGC ACTCCAGCCT GGGGGACAGA AAGAGACTCC 1600 GTCTCAAAAAAAAAAAAAAA AAAA 1624 267 amino acids amino acid unknown unknown unknown20 Met Gly Ser Ala Cys Ile Lys Val Thr Lys Tyr Phe 1 5 10 Leu Phe LeuPhe Asn Leu Ile Phe Phe Ile Leu Gly 15 20 Ala Val Ile Leu Gly Phe GlyVal Trp Ile Leu Ala 25 30 35 Asp Lys Ser Ser Phe Ile Ser Val Leu Gln ThrSer 40 45 Ser Ser Ser Leu Arg Met Gly Ala Tyr Val Phe Ile 50 55 60 GlyVal Gly Ala Val Thr Met Leu Met Gly Phe Leu 65 70 Gly Cys Ile Gly AlaVal Asn Glu Val Arg Cys Leu 75 80 Leu Gly Leu Tyr Phe Ala Phe Leu LeuLeu Ile Leu 85 90 95 Ile Ala Gln Val Thr Ala Gly Ala Leu Phe Tyr Phe 100105 Asn Met Gly Lys Leu Lys Gln Glu Met Gly Gly Ile 110 115 120 Val ThrGlu Leu Ile Arg Asp Tyr Asn Ser Ser Arg 125 130 Glu Asp Ser Leu Gln AspAla Trp Asp Tyr Val Gln 135 140 Ala Gln Val Lys Cys Cys Gly Trp Val SerPhe Tyr 145 150 155 Asn Trp Thr Asp Asn Ala Glu Leu Met Asn Arg Pro 160165 Glu Val Thr Tyr Pro Cys Ser Cys Glu Val Lys Gly 170 175 180 Glu GluAsp Asn Ser Leu Ser Val Arg Lys Gly Phe 185 190 Cys Glu Ala Pro Gly AsnArg Thr Gln Ser Gly Asn 195 200 His Pro Glu Asp Trp Pro Val Tyr Gln GluGly Cys 205 210 215 Met Glu Lys Val Gln Ala Trp Leu Gln Glu Asn Leu 220225 Gly Ile Ile Leu Gly Val Gly Val Gly Val Ala Ile 230 235 240 Ile GluLeu Leu Gly Met Val Leu Ser Ile Cys Leu 245 250 Cys Arg His Val His SerGlu Asp Tyr Ser Lys Val 255 260 Pro Lys Tyr 265

What is claimed is:
 1. A method for detecting the presence of malignantcancer in a subject, said method comprising: comparing the level of KAI1mRNA or KAI1 protein of said subject to the level of wild-type KAI1 mRNAor protein to determine the presence of a reduction of exression of KAI1mRNA-or protein, wherein said reduction of expression indicates thepresence of malignant cancer.
 2. The method of claim 1, wherein saidreduction of exression of KAI1 protein is detected by Western Blotting.3. The method of claim 1, wherein said reduction of exression of KAI1protein is detected by immunohistochemistry.
 4. The method of claim 1,wherein the wild-type KAI1 protein has an amino acid sequence accordingto SEQ ID NO:
 20. 5. A method for detecting the presence of malignantcancer in a subject, said method comprising: comparing the glycosylationof the KAI1 protein of said subject to the glycosylation of thewild-type KAI1 protein to determine the presence of a change inglycosylation, wherein loss of glycosylation indicates the presence ofmalignant cancer.
 6. The method of claim 5, wherein the change inglycosylation is detected by Western blotting.
 7. The method of claim 5,wherein the wild-type KAI1 protein has an amino acid sequence accordingto SEQ ID NO. 20.